HCV NS3 and NS5A: Biochemical Mechanisms and Biological Functions Abstract RNA metabolism requires the action of numerous ATP-dependent, molecular motor proteins that are believed to transport, remodel, and unwind secondary structures in RNA sequences. Many of these molecular motors are DEAD-box or closely related proteins. Positive strand RNA viruses such as the hepatitis C virus (HCV) require the activity of these proteins for RNA replication. Determination of the mechanism of these proteins is of fundamental importance to our understanding of viral replication as well as RNA metabolism in general. The long-term goal of this project is to determine the mechanism of RNA replication of HCV. The immediate focus of this proposal is on those proteins that bind and manipulate HCV RNA. Non-structural protein 3 (NS3) is an RNA motor protein (or helicase), that is necessary for HCV replication. Our data supports the hypothesis that NS3 exists in equilibrium between monomeric and oligomeric species and that its RNA unwinding activity increases with increasing oligomerization. We have devised new assays, reagents, and protocols that will allow us to test this hypothesis and to determine whether other HCV proteins such as NS5A can alleviate the need for oligomerization. We will examine helicase activity with high temporal and spatial resolution by using a new, chemical footprinting approach. We have studied the ATP hydrolysis cycle for NS3 and based on our preliminary data, we hypothesize that phosphate release limits the overall rate for RNA unwinding. We will examine protein motifs that are proposed to bind phosphate to test this hypothesis. Additionally, our work has identified NS5A as an RNA binding protein that also interacts directly with NS3. We will uncover the mechanism of NS5A RNA binding and its role in HCV replication through these studies. The interplay between the NS3 and NS5A will be examined in detail using new biochemical and biological approaches. In aim 1, we will Investigate the mechanism for unwinding and translocation of DNA and RNA by NS3 and relate the mechanism to ATP hydrolysis. In aim 2, we will determine the biochemical mechanism for RNA binding by NS5A and perform the first structure-function study of this protein. In aim 3, the molecular basis for NS3 interaction with NS5A will be examined by using a recently developed fluorescence assay along with our RNA unwinding, structural, and biological approaches. Specifc regions of protein-protien interactions will be identified by mass spectrometric methods.